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Department of Histology and Cell Biology, Umeå University, S-90187 Umeå, Sweden
Address all correspondence and requests for reprints to: Dr. Anders Edvell, Department of Histology and Cell Biology, Umeå University, S-901 87 Umeå, Sweden. E-mail: ansedl97{at}student.umu.se
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Abstract |
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Introduction |
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The obese hyperglycemic mouse (ob/ob) is a model for increased ß-cell proliferation. ob/ob mice are characterized by hyperphagia, obesity, hyperinsulinemia, and hyperglycemia (5, 6). A previous study found increased blood glucose levels and increased ß-cell proliferation by 3 weeks of age (7).
We have now dissected the course of events in greater detail and find that increased islet cell proliferation precedes increased blood glucose by 2 days in young obese mice, suggesting that increased ß-cell proliferation is a consequence of increased demand for insulin rather than of increased blood glucose levels. Furthermore, when insulin is administered sc in young obese animals in doses sufficient to lower the endogenous demand for insulin but not sufficient to lower blood glucose levels, the ß-cell proliferation rate is reduced to the levels found in lean animals. We therefore wanted to look for possible initiators of islet growth, other than glucose, that are related to increased food intake and insulin release.
We choose to study the effect of glucagon like peptide-1 (GLP-1) because it is recognized as an important gut-derived stimulator of insulin release that is released early in relation to meals (8). C Peptide stimulates Na+,K+-adenosine triphosphatase (Na+,K+-ATPase) activity in renal tubuli (9), and it has been reported that it may do so also in pancreatic islets (10). Increased Na+,K+-ATPase activity is coupled to increased proliferation in other cell systems (11, 12). Because C-peptide is released together with insulin, we found it intriguing to examine its effect on proliferation.
We found that both the incretin GLP-1 and C peptide stimulated islet growth. This suggests that a number of factors other than blood glucose per se may be important in the initiation of the increased islet cell proliferation rate in response to increased metabolic demand.
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Materials and Methods |
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Injection of animals
On day 18, obese mice were separated from their lean
littermates. In the evening of day 18, during the morning, midday, and
evening of day 19, and during the morning of day 20, lean mice
were injected with 13, 27, 40, and 53 nmol/kg BW GLP-1
(Sigma Chemical Co., St. Louis, MO); 1.8, 5.4, and 16
nmol/kg synthetic human C peptide (Sigma Chemical Co.);
400 nmol/kg vasoactive intestinal polypeptide (VIP; Sigma Chemical Co.); and 375 µmol/kg
L-5-hydroxytryptophan (L-5-HTP; Sigma Chemical Co.). The injections were given sc. These or lower
doses have previously been shown to have metabolic effects such as
lowered plasma glucose and increased insulin release
(GLP-1) (13, 14), increased glucose utilization (C
peptide) (15), hypoglycemia in rats (VIP) (16), and reduced subsequent
insulin release in injected animals (L-5-HTP) (17). The
insulin given was Insulatard (100 U/ml; Novo Nordisk A/S,
Copenhagen, Denmark); 4.1 U/kg (24 nmol/kg) were given on day 18, and
6.2 U/kg (36 nmol/kg) were given on days 19–20. Insulin was
administered twice daily (morning and early evening). On day 20 the
mice were injected with 5-bromo-2'-deoxyuridine (BrdU; 300 mg/kg, ip)
2 h before death. Blood samples for glucose and insulin
measurements were collected and analyzed by immunohistochemical methods
as described below. Jejunal and kidney tissue were also fixed for
studies of possible proliferative effects after treatment with
GLP-1 and C peptide. Pancreases from insulin-injected
animals and controls were investigated as described in the culture
experiments. In experiments performed in rats, 5.4 nmol/kg C peptide
and the same number of injections as in mice were given.
Immunohistochemical staining
Pancreas was removed on day 20 (see above). It was fixed in
Bouin’s solution, dehydrated, embedded in paraffin, and cut
transversely with a microtome. Every 20–30th slice was placed on a
microscope slide. After removal of paraffin, the slides were incubated
for 10 min in Tris buffer supplemented with 30%
H2O2 to reduce endogenous peroxidase activity.
The slides were then rinsed in Tris buffer (60.6 g Tris, 79.0 g
NaCl, 10 liters H2O, and 1.0 M HCl in
sufficient amounts to obtain pH 7.6). Tris was purchased from
Boehringer Mannheim (Mannheim, Germany). After this the slides were
incubated in 10 mg/ml BSA to reduce background staining. The slides
were incubated overnight at room temperature in buffer containing
monoclonal anti-BrdU antibody (Amersham, Aylesbury, UK), diluted 1:7 in
Tris. After this the slides were rinsed and incubated for 30 min with
antimouse IgG antibody diluted 1:25, supplemented with 15% normal
rabbit serum. After rinsing, the slides were incubated for 30 min with
alkaline phosphatase-antialkaline phosphatase complex diluted 1:50.
Alkaline phosphatase activity was finally revealed by incubation for 30
min in 5-bromo-chloro-3-indolyl phosphate and nitro blue tetrazolium,
supplemented with levamisole diluted 1:25. Chemicals for immune
histochemistry were obtained from Dako Corp.
(Copenhagen, Denmark). After rinsing in distilled
H2O, slides were counterstained for 10 min with calcium
red. Finally, the slides were dehydrated and mounted. With this
technique labeled nuclei are stained dark blue, and nonlabeled nuclei
are stained pink.
Insulin-containing cells were identified as follows. After removal of the paraffin, slides were incubated for 90 min at room temperature with a primary antibody directed against insulin (Eurodiagnostica, Malmo, Sweden) diluted 1:200 in Tris. The slides were rinsed, and a secondary antibody (Dako Corp.), marked with biotin and diluted 1:200, was added for 30 min. The slides were rinsed again and incubated for 30 min with avidin-biotin complex (Dako Corp.). Finally, the slides were stained with diaminobenzidine solution (Dako Corp.) for 5 min. After this the procedure for BrdU staining continued as described above. With this technique, labeled nuclei stained dark blue, and cytoplasma containing insulin stained light brown. To determine the labeling index, slides were coded and examined under an oil immersion lens (total magnification, x1000). In each experiment at least 2500 islet cells/group were counted.
Culture experiments
On day 20, lean and obese mice were killed. Blood was collected
for analysis of glucose and insulin. Pancreas was dissected out, and
islets were collagenase isolated as described previously (7).
ob/ob mouse islets were counted and put in cell
culture suspension dishes from Sarstedt (Landskrona, Sweden). RPMI
medium (Flow Laboratories Ltd., Irvine, UK) was used with 11.1
mM glucose together with 10% heat-inactivated (60 C, 60
min) serum from lean or obese mice. In experiments performed on islets
from lean mice, FCS was used. To this medium 13.5 or 68
µM GLP-1, 9.2 or 27.6 µM C
peptide, or 23 µM L-5-HTP was added. It has
been reported that 100 nM GLP-1 stimulates
insulin secretion in islet culture (13), and that C peptide stimulates
renal tubular Na+K+-ATPase activity in a dose
range of 100 nM to 1 µM (9).
Culture dishes were incubated in a cell culture chamber (CO2-Auto-Zero, Heraeus, Hanau, Germany) at 37 C with 5% CO2 in air. After 30 ± 3 h, islets were rinsed and incubated for another 18 ± 2 h in RPMI medium containing 9 mM hydroxyurea to synchronize cell division cycles (18). After that, islets were put in fresh culture medium for 5 h. Islets were then preincubated for 30 min in Hanks’ buffer at 37 C and incubated for 2 h in Krebs-Ringer bicarbonate HEPES solution containing 115 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 2.56 mM CaCl2, 20 mM NaHCO3, 3 mM D-glucose, and 20 mM HEPES, pH 7.40. The medium also contained 1 mg/ml BSA (Miles Laboratories, Stoke Poges, UK) and 1.5 µCi/ml [3H]thymidine (126 gigabecquerels/mmol). [3H]Thymidine was obtained from the Radiochemical Center, Amersham (Aylesbury, UK). Islets were rinsed in Hanks’ solution with 1 mg/ml albumin several times and put in Bouin’s solution. Then islets were dehydrated, embedded in paraffin, sectioned, and mounted on microscope slides. After the removal of paraffin, the sections were dipped in Kodak NTB-2 emulsion (Eastman Kodak, Inc., Rochester, NY) and exposed for 1 week at 6 C. The autoradiographs were developed for 5 min in Kodak D-19 and fixed for 10 min in Stena Scanfors (Stockholm, Sweden) solution. The sections were dried overnight and were counterstained with hematoxylin and eosin. Labeled cells were counted as described above.
Glucose and insulin analysis
All blood samples were obtained from the retroorbital plexa. To
analyze blood glucose, an Accutrend alpha (Boehringer Mannheim GmbH,
Mannheim, Germany) was used. Blood was collected with a capillary
blood-collecting tube (KEBO Laboratory, Stockholm, Sweden) and was
taken between 0800–1200 h and between 1800–2200 h. In 19-day-old
animals, blood glucose samples were also obtained 1, 2, 3, 5, and
7 h after insulin injection. To calibrate blood glucose values
obtained by this method, originally developed for human blood, a method
using the luciferin/luciferase system and a liquid scintillation
spectrometer was used (19). Luciferin and luciferase were obtained from
Boehringer Mannheim. Serum insulin samples were assayed by RIA, using
crystalline mouse insulin as a standard. 125I was obtained
from Eurodiagnostica (Malmo, Sweden).
Statistical methods
Statistical analysis is made using Student’s t test
for paired or independent data.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. This is at a time when blood glucose
values are not different from those in lean littermates. A difference
in blood glucose values between obese and lean littermates was observed
on day 22 (7.0 ± 0.7 and 4.9 ± 0.4 mM,
respectively).
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shows that there was no persistent
effect on blood glucose levels in insulin-treated animals over the
experimental period. There was a tendency toward slightly lower blood
glucose levels during the first 2 h after insulin injection (Fig. 2B) although it did not reach statistical significance with the numbers
of animals tested. The proliferation rate was lower in insulin-injected
obese mice compared with saline-injected obese controls from the same
litter. This is shown in Fig. 3.
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).
GLP-1 also stimulated islet cell labeling in a
dose-dependent manner (Fig. 4). When VIP was administered, no change in
labeling was observed (1.38 ± 0.28% vs. 1.47 ±
0.12% in control mice; n = 5 and 33). In animals treated with
L-5-HTP, there was a decreased islet cell labeling index
compared with that in saline-injected mice (1.13 ± 0.23%
vs. 1.90 ± 0.32%; P < 0.05; n =
7 for both groups). None of these treatments affected the blood glucose
values (data not shown).
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To determine whether the different treatments affected serum insulin levels, insulin was measured in mice given 16 nmol/kg C peptide, and mice given 53 nmol/kg GLP-1, and control mice. No difference was detected when comparing the groups [5.5 ± 0.8 ng/ml (n = 7) in C peptide-treated mice, 5.7 ± 0.7 ng/ml (n = 7) in GLP-1-treated mice, and 4.5 ± 0.33 ng/ml (n = 7) in the control group].
Slides were stained for insulin to compare the labeling index of ß-cells vs. non-ß-cells after C peptide and GLP-1 injections. Pancreatic sections from five or six animals in each group were observed. In control animals, 79 of a total of 95 (83%) BrdU-positive cells also stained for insulin. Corresponding values were 119 of 135 (88%) in mice injected with C peptide and 89 of 99 (90%) in mice injected with GLP-1. There was no statistically significant difference between the proportion of insulin staining cells in these groups.
To obtain an estimate of the effect on proliferation in tissues other than pancreatic islets, the BrdU labeling index was measured in the exocrine pancreas, in mouse kidney, and in jejunal villi. The labeling index in exocrine pancreatic cells was 2.0 ± 0.5% (n = 9) in control mice, 2.5 ± 0.4% (n = 7) in mice injected with C peptide, and 2.1 ± 0.6% (n = 5) in mice injected with GLP-1; none of the differences was significant. The labeling index in kidney cortex was 3.3 ± 0.3% (n = 5) in control mice and 3.7 ± 0.3% (n = 5; P = NS) and 3.1 ± 0.5% (n = 5; P = NS) in GLP-1- and C peptide-treated mice, respectively. In the distal part of jejunal villi, the labeling index was 2.4 ± 0.2% (n = 5) in control mice, 2.5 ± 0.2% (n = 5; P = NS) and 1.9 ± 0.2% (n = 5; P = NS) in GLP-1- and C peptide-treated mice, respectively.
Culture experiments
To test the islet cell growth-promoting effect of ob/ob
mouse serum, islets from obese or lean mice were cultured for 2 days in
medium supplemented with serum from young obese or young lean animals.
The labeling index was 2.5 ± 0.4% (n = 6) in obese islets
and 1.9 ± 0.4% (n = 7; P = NS) in lean
mouse islets after culture in medium containing obese mouse serum. The
corresponding values in islets cultured with lean mouse serum were
1.9 ± 0.4% and 1.9 ± 0.8% (n = 7), which were not
significantly different compared with the proliferation rate in islets
cultured in serum from obese mice. The presence of GLP-1
or L-5-HTP during culture did not affect the islet cell
labeling rate in islets from lean mice (Fig. 5). However, when islets were cultured in
the presence of C peptide there was an elevated islet cell
labeling index (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A large number of growth factors and growth-stimulating peptides are expressed in or have stimulatory effects in the growing islet (20). Less is known about the initial inductive signals that control ß-cell proliferation. We wanted to identify factors that are related to increased islet functional demand and that can have a direct triggering effect on ß-cell proliferation. This response can then be enhanced, modified, and perhaps mediated by a number of other factors. We chose to study the effects of GLP-1. GLP-1 is recognized as an important gut-derived stimulator of insulin release, released early in relation to meals (8). GLP-1 can also induce glucose responsiveness in fetal islets (21). Obese hyperglycemic mice have a large food intake, which could stimulate GLP-1 release. Incretin-induced insulin release could be one explanation for the high insulin levels detected in young obese mice. The finding that GLP-1 stimulated islet growth in 20-day-old mice suggests that gut factors promote islet proliferation. Earlier studies have demonstrated that GLP-2 is involved in mitotic actions in the intestinal tract (22). To our knowledge there are no earlier studies that have investigated the proliferative effects of GLP-1 in the pancreas or pancreatic islets. The effects of GLP-1 may be indirect because of the lack of effect on isolated islets of GLP-1 concentrations known to stimulate insulin release during islet culture (13).
Biogenic monoamines such as 5-hydroxytryptamine and dopamine are present in higher amounts in the islets of young animals (23) that also show a larger proliferative capacity. Mouse ß-cells accumulate 5-hydroxytryptamine when they are pretreated with the 5-hydroxytryptamine precursor L-5-HTP (17), and this inhibits insulin secretion, in contrast to the stimulatory effects observed in acute experiments with L-5-HTP (24). We found a small inhibitory effect of L-5-HTP pretreatment on the islet cell proliferation rate. The L-5-HTP dose was chosen because it is known to inhibit insulin release in subsequent experiments (17), but more experiments are needed to more precisely evaluate the effect of L-5-HTP on islet cell proliferation. L-5-HTP pretreatment inhibited islet proliferation despite normal blood glucose. This suggests that insulin secretion per se is important for triggering of an increased mitotic rate. Further support for this came from experiments with insulin injections (discussed below) and C peptide. VIP had no effect on the islet cell labeling index at a concentration known to induce hypoglycemia in rats. Although data on other doses are lacking, this suggests no strong stimulatory effect of VIP on islet cell proliferation.
C peptide is coreleased with insulin in equimolar concentrations, probably leading to increased C peptide levels in obese hyperglycemic mice. It has been suggested that C peptide inhibits insulin release in rats (25, 26), but this was not confirmed in human studies (27). C Peptide also stimulates Na+,K+-ATPase activity in renal tubuli (9), and activation of the Na+,K+-ATPase is correlated with increased proliferation in lymphocytes (11) and astrocytes (12). Therefore, it is possible that C peptide can affect ß-cell intracellular events, although no receptor has been identified. A stimulating effect on the islet cell proliferation rate, perhaps concomitant with inhibition of insulin secretion, suggests a complex role for C peptide in islet physiology. A highly purified synthetic human C peptide was used (>95%) to avoid functional interference of contaminants in the C peptide preparation.
When young rats were given the same dose of C peptide as mice, no effect on BrdU labeling was observed. There are species variations in C peptide molecular structure. Further studies are therefore needed to clarify whether this lack of a growth-stimulating effect of C peptide in islets from young rats reflects a qualitative species difference.
Eighty-eight to 90% of the labeled islet cells also stained for insulin, suggesting that C peptide and GLP-1 stimulate the proliferation of ß-cells in islets from young mice. The finding that C peptide and GLP-1 had no effect on the BrdU labeling of exocrine pancreatic cells or on cells from kidney cortex or jejunal villi suggests some specificity for the growth-promoting effect on the islets.
Swenne found that neonatal rat islets cultured in serum from lean or obese mice had a higher DNA synthesis than islets cultured in FCS (28). The effect was not dependent on the glucose concentration in the medium (28). We now find that the islet cell proliferation rate is also the same in cultured obese and lean mouse islets when cultured in medium containing sera from 20-day-old obese animals. This suggests that proliferation in vivo is dependent on local factors (29) or on a serum component that is not present in amounts high enough to trigger islet cell proliferation in addition to the effect of 11.1 mM glucose. The findings strengthen the view that ob/ob mouse islets are essentially normal and that islet growth, with up to 10 times larger islets than those in lean mice (30), is an adaptation to the obese syndrome.
There is good evidence that ob/ob mice are under increased metabolic demand for insulin secretion in the immediate postweaning period. Serum insulin levels are already high on day 17 (31). Because of the increased insulin secretion, blood glucose levels remain normal for a few days, and the animals do not become hyperglycemic until day 22. However, increased islet cell proliferation is observed on day 20. This suggests that the signal for islet cell proliferation is related to a long standing demand for increased insulin secretion. It is probably not a sign of the ß-cells failing to respond to the demand with ensuing hyperglycemia.
How, then, is increased proliferation related to the glucose signal for insulin release? Islets from young obese mice may be more sensitive to cholinergic stimulation of insulin release than their lean littermates (32). An increased sensitivity for glucose could also explain why insulin release and islet proliferation are enhanced in young obese mouse islets despite similar blood sugar values as those in lean mice. An increased islet cell proliferation before hyperglycemia is also seen in BB rats (33) and db/db mice (34). However, islet cell proliferation in obese mice treated with a low dose of insulin was reduced despite very small, if any, effects on blood glucose. These findings speak against the idea that the increased islet proliferation in obese mice is caused by an increased sensitivity for glucose. We found a proliferative effect of added C peptide, and a decreased islet cell proliferation after administration of exogenous insulin. Exogenous insulin reduces the demand for endogenous insulin secretion and thus reduces C peptide release. This supports the view that proliferation is related to the actual insulin release rather than the strength of metabolic stimulus measured as blood sugar values.
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Footnotes |
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Received April 20, 1998.
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) and obese (
) mice. Values are the
mean ± SE (n = 5–10 in A; n = 25–52 for
days 18–21 and 6–16 for days 22–28 in B). *, P
< 0.05 when comparing obese and lean mice of same age, using
Student’s t test for independent data.
) were
injected with saline. Data are presented as the mean ±
SE (n = 4–7 on day 17 and n = 6–18 on days
18–20). No significant differences between groups were observed, using
Student’s t test for paired or independent data. B, A
more detailed presentation of blood glucose values obtained during day
19. The first samples were taken immediately before insulin injection
at 1000 h. The mean ± SE for six to eight
observations are shown.
